Using a cohort of more than 250 subjects with a wide variety of eosinophilic disorders, ranging from benign eosinophilia to eosinophilic leukemia, we have continued to look for new markers that distinguish between variants of hypereosinophilic syndrome (HES). Assessment of serum and surface IL-5 receptor levels in a large cohort of patients with eosinophilia and/or mastocytosis suggests that regulation of this receptor occurs in vivo (Wilson et al J Allergy Clin Immunol 2011). Serum levels of the receptor were correlated with eosinophilia, serum levels of IL-5 and IL-13 and markers of eosinophil activation. Interestingly, serum receptor levels were also elevated in patients with systemic mastocytosis without eosinophilia and correlated with both serum tryptase and surface markers of eosinophil activation. These findings may have important implications in the setting of recently developed therapeutic agents targeting IL-5 and its receptor. In collaboration with Bruce Bochner, we have begun to characterize the expression and regulation of siglec-8, an inhibitory marker on eosinophils, in our cohort of subjects with HES (Na et al. J Immunol Methods 2011). Surface expression of sigelc-8 has been confirmed on eosinophils from subjects with a wide variety of eosinophilic disorders and an assay has been developed to detect the soluble form of siglec-8 in human serum. Siglec-8 is a potential therapeutic target in eosinophilic disease. Thus, the presence and levels of soluble receptor could interfere with therapeutic efficacy. Additional studies are ongoing with siglec-8 and several other eosinophil surface markers of interest. The utility of a variety of biomarkers in distinguishing between subgroups of eosinophilic subjects with biopsy-proven ANCA-negative Churg-Strauss vasculitis (CSS), suspected ANCA-negative CSS and HES with and without asthma and sinusitis was explored (Khoury et al. Allergy 2012). Although serum levels of a number of markers were significantly different in subjects with HES without asthma or sinusitis as compared to subjects in the other eosinophilic groups and in all eosinophilic subjects as compared to normal controls, no biomarkers were able to distinguish between ANCA-negative definite or suspected CSS and HES with asthma and/or sinusitis. These data suggest that ANCA-negative CSS and HES with asthma and/or sinusitis may share a common pathogenesis supporting the use of therapies targeting eosinophilia in ANCA-negative CSS. We have previously described a family with an autosomal dominant variant of hypereosinophilic syndrome that maps to chromosome 5q31-33. Exhaustive attempts to identify the gene responsible for this condition, including candidate gene sequencing, array CGHY, expression analysis, 454 sequencing of the region upstream of IL-5, in collaboration with John Rioux and Jan Cools, have been unsuccessful to date. Whole genome sequencing of DNA from two family members has been completed and a candidate polymorphism has been identified that is present in only affected family members. Studies are underway to confirm the role of this polymorphism in the etiology of the eosinophilia. We continue to follow affected family members for development of clinical manifestations. To date, there has been no progression of disease and markers of eosinophil activation have remained stable. In the past year, we have also continued to characterize several novel subgroups of patients with eosinophilia. We studied four patients with episodic eosinophilia and angioedema and demonstrated that multiple lineages, including neutrophils, eosinophils and platelets, cycle with a 25-30 day periodicity. Eosinophil cycling is preceded by elevations in eosinophil related cytokines, including IL-5, GM-CSF and eotaxin, and followed by rises in inflammatory markers. Unlike cyclic neutropenia, mutations in ELANE do not appear to be involved. We have also identified 7 subjects with marked eosinophilia (>1,500/mm3) of >5 years duration and no evidence of clinical manifestations despite not being treated. Comparison of these subjects to patients with hypereosinophilic syndrome reveals that they are a heterogeneous group, but have little evidence of eosinophil activation. A multicenter study is being organized to examine and compare subjects who are asymptomatic despite marked eosinophilia to those who present without symptoms but go on to develop clinical disease. Finally, we have initiated a new clinical protocol to determine whether the acute response to a single dose of prednisone can predict the dose ultimately needed to control eosinophilia and clinical manifestations of HES and to explore the mechanisms of glucocorticoid resistance in HES. The effects of novel therapies for the treatment of hypereosinophilic syndromes, including monoclonal antibodies to IL-5 and the tyrosine kinase inhibitor, imatinib, remain a primary focus. Long-term (5 year) followup of subjects enrolled on a placebo-controlled study of the anti-IL-5 antibody, mepolizumab has just been completed. The drug continued to show excellent efficacy in the majority of subjects and was not associated with toxicity or the development of neutralizing antibodies (Roufosse et al. 2012 J Allergy Clin Immunol). We have also begun to explore new therapeutic approaches for the treatment of refractory L-HES in collaboration with Dr. Dunleavy (NCI)and several novel targeted therapies with industry collaborators. Treatment of the filarial infection loiasis with diethylcarbamazine (DEC) can be associated with severe adverse events, including death, that are associated with a dramatic rise in eosinophilia and are positively correlated with the number of circulating microfilariae in the blood. In the past year, we have expanded our studies of monoclonal antibodies targeting IL-5 to explore the role of eosinophilia in post-treatment reactions following DEC treatment of loiasis. A double-blind, placebo-controlled study of the anti-IL-5 antibody, reslizumab, to prevent post-treatment side effects of DEC in patients with Loa loa infection is currently underway. Because similar side effects have been seen in patients with loiasis participating in mass drug treatment campaigns with the drug, ivermectin, we have also initiated a clinical study in Cameroon to compare clinical and immunological post-treatment effects of DEC and ivermectin in patients with low levels of circulating Loa loa microfilariae. The clinical portion of the study has been completed and data analysis is underway.